Project Summary Satellite cells are muscle-specific stem cells that are responsible for skeletal muscle growth and regeneration. The myogenic regulatory factors (MRFs) MYOD and MYF5 are essential for muscle lineage determination in the embryo and are induced in activated satellite cells as an early response to muscle injury. Recent gene- targeting studies using a new MyoD conditional knockout allele (MyoDcKO) showed that either MyoD or Myf5 is essential for muscle regeneration; satellite cells lacking both genes (dKO) accumulate in injured muscle but are unable to undergo myogenic differentiation. In this proposal, new genetic tools and strategies are used to determine the functions of MyoD and Myf5 in satellite cell development, lineage determination, differentiation and self-renewal. In addition, transcriptional control mechanisms that regulate MyoD expression in satellite cells and during embryogenesis are interrogated. In Aim 1, cell type identification by immunofluorescence and single-cell RNA sequencing (scRNA-seq) will establish whether dKO satellite cells adopt non-myogenic cell fates, the extent to which they retain myogenic programming, and their capacity for self-renewal. Experiments will also distinguish cell-autonomous and non-autonomous effects of MRF deficiency. Aim 2 will utilize RNA- seq to define the transcriptome of mutant satellite cells in uninjured and injured skeletal muscle, which will identify direct and indirect transcriptional targets of MYOD and MYF5 as well as regulatory pathways and cellular processes impacted by the loss of these MRFs. In addition, Pro-seq (genome-wide Precision Run-On) analyses will quantify changes in active gene transcription, will identify candidate genes regulated by promoter- proximal polymerase pausing, and will identify potential enhancer targets of MYOD and MYF5. Aim 3 will determine whether the function of MyoD or Myf5 is required for satellite cell development by producing dKO satellite cell precursors at embryonic, fetal and neonatal stages and testing their capacity to generate adult satellite cells, as assessed by molecular and anatomical criteria. Recent data demonstrate that the only enhancer elements known to regulate MyoD expression (the core enhancer and distal regulatory region) are not necessary for MyoD transcription during embryogenesis or in satellite cells. Aim 4 will utilize transfection, transgenic and CRISPR-based knockout methodologies to define the regulatory functions of novel putative enhancer elements identified by PRO-seq and bioinformatic analyses. The proposed research will contribute significantly to an understanding of fundamental gene regulatory mechanisms that control satellite cell stem cell functions and may lead to the development of new cell types and strategies for cell-based therapies for muscle degenerative diseases.